Extracellular Norepinephrine Clearance by the Norepinephrine Transporter Is Required for Skeletal Homeostasis

Changes in bone remodeling induced by pharmacological and genetic manipulation of β-adrenergic receptor (βAR) signaling in osteoblasts support a role of sympathetic nerves in the regulation of bone remodeling. However, the contribution of endogenous sympathetic outflow and nerve-derived norepinephrine (NE) to bone remodeling under pathophysiological conditions remains unclear. We show here that differentiated osteoblasts, like neurons, express the norepinephrine transporter (NET), exhibit specific NE uptake activity via NET and can catabolize, but not generate, NE. Pharmacological blockade of NE transport by reboxetine induced bone loss in WT mice. Similarly, lack of NE reuptake in norepinephrine transporter (Net)-deficient mice led to reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and mechanical properties associated with low sympathetic outflow and high plasma NE levels. Last, daily sympathetic activation induced by mild chronic stress was unable to induce bone loss, unless NET activity was blocked. These findings indicate that the control of endogenous NE release and reuptake by presynaptic neurons and osteoblasts is an important component of the complex homeostatic machinery by which the sympathetic nervous system controls bone remodeling. These findings also suggest that drugs antagonizing NET activity, used for the treatment of hyperactivity disorders, may have deleterious effects on bone accrual.     

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

A small tripeptide AFA undergoes two state cooperative conformational transitions: implications for conformational biases in unfolded states

It is important to understand the conformational biases that are present in unfolded states to understand protein folding. In this context, it is surprising that even a short tripeptide like AFA samples folded/ordered conformation as demonstrated recently by NMR experiments of the peptide in aqueous solution at 280 K. In this paper, we present molecular dynamics simulation of the peptide in explicit water using OPLS-AA/L all-atom force field. The results are in overall agreement with NMR results and provide some further insights. The peptide samples turn and extended conformational forms corresponding to minima in free energy landscape. Frequent transitions between the minima are observed due to modest free energy barriers. The turn conformation seems to be stabilized by hydrophobic interactions and possibly by bridging water molecules between backbone donors and acceptors. Thus the peptide does not sample conformations randomly, but samples well defined conformations. The peptide served as a model for folding-unfolding equilibrium in the context of peptide folding. Further, implications for drug design are also discussed.     

Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our?study implicates five networks of kinases that?regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in?vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.     

A shorter peptide model from staphylococcal nuclease for the folding-unfolding equilibrium of a beta-hairpin shows that unfolded state has significant contribution from compact conformational states

It is important to understand the conformational features of the unfolded state in equilibrium with folded state under physiological conditions. In this paper, we consider a short peptide model LMYKGQPM from staphylococcal nuclease to model the conformational equilibrium between a hairpin conformation and its unfolded state using molecular dynamics simulation under NVT conditions at 300K using GROMOS96 force field. The free energy landscape has overall funnel-like shape with hairpin conformations sampling the minima. The "unfolded" state has a higher free energy of approximately 12kJ/mol with respect to native hairpin minimum and occupies a plateau region. We find that the unfolded state has significant contributions from compact conformations. Many of these conformations have hairpin-like topology. Further, these compact conformational forms are stabilized by hydrophobic interactions. Conversion between native and non-native hairpins occurs via unfolded states. Frequent conversions between folded and unfolded hairpins are observed with single exponential kinetics. We compare our results with the emerging picture of unfolded state from both experimental and theoretical studies.     

Loop propensity of the sequence YKGQP from staphylococcal nuclease: implications for the folding of nuclease.

Recently we performed molecular dynamics (MD) simulations on the folding of the hairpin peptide DTVKLMYKGQPMTFR from staphylococcal nuclease in explicit water. We found that the peptide folds into a hairpin conformation with native and nonnative hydrogen-bonding patterns. In all the folding events observed in the folding of the hairpin peptide, loop formation involving the region YKGQP was an important event. In order to trace the origins of the loop propensity of the sequence YKGQP, we performed MD simulations on the sequence starting from extended, polyproline II and native type I' turn conformations for a total simulation length of 300 ns, using the GROMOS96 force field under constant volume and temperature (NVT) conditions. The free-energy landscape of the peptide YKGQP shows minima corresponding to loop conformation with Tyr and Pro side-chain association, turn and extended conformational forms, with modest free-energy barriers separating the minima. To elucidate the role of Gly in facilitating loop formation, we also performed MD simulations of the mutated peptide YKAQP (Gly --> Ala mutation) under similar conditions starting from polyproline II conformation for 100 ns. Two minima corresponding to bend/turn and extended conformations were observed in the free-energy landscape for the peptide YKAQP. The free-energy barrier between the minima in the free-energy landscape of the peptide YKAQP was also modest. Loop conformation is largely sampled by the YKGQP peptide, while extended conformation is largely sampled by the YKAQP peptide. We also explain why the YKGQP sequence samples type II turn conformation in these simulations, whereas the sequence as part of the hairpin peptide DTVKLMYKGQPMTFR samples type I' turn conformation both in the X-ray crystal structure and in our earlier simulations on the folding of the hairpin peptide. We discuss the implications of our results to the folding of the staphylococcal nuclease.     

Energetics of galactose- and glucose-aromatic amino acid interactions: implications for binding in galactose-specific proteins

An aromatic amino acid is present in the binding site of a number of sugar binding proteins. The interaction of the saccharide with the aromatic residue is determined by their relative position as well as orientation. The position-orientation of the saccharide relative to the aromatic residue was found to vary in different sugar-binding proteins. In the present study, interaction energies of the complexes of galactose (Gal) and of glucose (Glc) with aromatic residue analogs have been calculated by ab initio density functional (U-B3LYP/ 6-31G**) theory. The position-orientations of the saccharide with respect to the aromatic residue observed in various Gal-, Glc-, and mannose-protein complexes were chosen for the interaction energy calculations. The results of these calculations show that galactose can interact with the aromatic residue with similar interaction energies in a number of position-orientations. The interaction energy of Gal-aromatic residue analog complex in position-orientations observed for the bound saccharide in Glc/Man-protein complexes is comparable to the Glc-aromatic residue analog complex in the same position-orientation. In contrast, there is a large variation in interaction energies of complexes of Glc- and of Gal- with the aromatic residue analog in position-orientations observed in Gal-protein complexes. Furthermore, the conformation wherein the O6 atom is away from the aromatic residue is preferred for the exocyclic -CH2OH group in Gal-aromatic residue analog complexes. The implications of these results for saccharide binding in Gal-specific proteins and the possible role of the aromatic amino acid to ensure proper positioning and orientation of galactose in the binding site have been discussed.     

Recombinant human epidermal growth factor inclusion body solubilization and refolding at large scale using expanded-bed adsorption chromatography from Escherichia coli

Amongst the various endogenous growth factors, epidermal growth factor (EGF) plays an important role in normal wound healing of tissue such as skin, cornea and gastrointestinal tract. Various studies have proved that supplementing recombinant human EGF (rhEGF) results in significant augmentation of wound healing. In the present work, a high level expression system with poly-arginine sequences was used for the production of recombinant human EGF (rhEGF) as inclusion bodies. The inclusion bodies were solubilized and the protein was refolded by using expanded-bed adsorption chromatography. The renatured protein was digested with appropriate concentration of trypsin and subsequently the digested rhEGF is purified by passing through ion-exchange chromatography (Toyopearl-SP) to obtain a biologically active protein. This process is the shortest process with reduced number of steps of purification, eliminates the usage of preparative reversed phase HPLC (RP-HPLC) for final purification, which is an expensive technique. The purified protein was analyzed by RP-HPLC, showing a purity >99% and size exclusion chromatography profile shows that there are minimal aggregates, with 99% renatured active protein. The purified rhEGF showed a specific activity of 5 × 10⁵ IU/mg protein, in comparison with NIBSC standard (1st International Standard of rDNA-derived EGF, Code 91/530). The process has been successfully adopted at 100 L fermentation scale and the rhEGF based formulation has been commercialized with brand name REGEN D, with excellent clinical results.